There is substantial evidence that an effective HIV vaccine to combat the AIDS epidemic must target the tremendous diversity of the HIV envelope glycoprotein. Our contention is that a multiple-envelope vaccine that elicits immune responses to a diversity of envelope epitopes will generate broad protection against naturally occurring viral isolates. Support for this hypothesis is provided by preliminary clinical trials of our PolyEnve1 vaccine, which presents 23 different envelopes in the form of recombinant vaccinia viruses. The development of approaches to enhancing the effectiveness of PolyEnv1 is now a primary goal. Our preliminary studies in a mouse model strongly supports the comprehensive evaluation of a multi- step strategy in which envelopes are first administered as DNA, than as recombinant vaccinia viruses, and finally as purified protein (i.e., a D-V-P regimen incorporating PolyEnv1, 23 matching envelopes in the form of DNA, and a selected subset of 6 envelopes as purified protein. Monoclonal antibodies to facilitate the typing of immune responses will be generated to the purified proteins, the diversity of responses to the protein component of the DNA component alone will be evaluated, and the magnitude and diversity of the response to the complete D-V-P regimen will be assessed. The second part of this project will address the phenomenon of "original antigenic sin", whereby boosting immunizations with variant molecules serve only to enhance antibody responses to epitopes that are shared with the priming antigen, and are less effective in eliciting responses to novel epitopes. Experiments will be conducted to test the hypothesis that the D-V- P vaccination strategy will circumvent "original antigen sin" and generate an optimally diverse antibody response. The studies described in this project are an important step towards realizing the potential of multiple-envelope HIV vaccines.